New Polyprenylated Acylphloroglucinol Derivatives and Xanthones From Hypericum wilsonii

Four new polyprenylated acylphloroglucinol derivatives, hyperwilone A-D (1–4), and two new xanthones, wilsonxanthone A (5) and wilsonxanthone B (6), together with eight known compounds were isolated from the aerial parts of Hypericum wilsonii. Their structures were expounded by comprehensive analysis of the 1D and 2D NMR spectra and HRESIMS. The relative configurations and absolute configurations of 1-6 were determined by NMR calculations and comparing their experimental and computed ECD data. All compounds were evaluated for GLUT4 translocation effects in L6 myotubes. Compound 5 showed the strongest GLUT4 translocation effects with 2.57 folds at a concentration of 30 μg/ml.


INTRODUCTION
Diabetes is a common chronic disease caused by the combined action of genetic and environmental factors. Type 2 diabetes (T2DM) featured with insulin resistance is the most prevalent type of diabetes in the population, accounting for around 90% of all diabetes cases (Zimmet et al., 2001). A large amount of human and financial resources have been invested globally in the fight against T2DM and its related complicating disease (Ha do et al., 2009). This problem is especially widespread in China due to the rapid improvement of people's quality of life and lack of exercise (Yang et al., 2017a). Although current antidiabetic treatment strategies have proven to be quiet effective, there are still some questions about tolerability and mechanism-based side effects (Cramer et al., 2008). Therefore, the development of more safer and effective antidiabetic drugs is in line with the actual needs of people today. Glucose transporter 4 (GLUT4) has a crucial role in systemic glucose homeostasis, which is one of the most potential target in the development of an antidiabetic drug (Leto and Saltiel, 2012). An increasing body of evidence suggests the enhancement of GLUT4's translocation can revive insulin resistance; therefore, it hopefully leads to the exploitation of the new antidiabetic medicine (Zhang et al., 2007;Naresh et al., 2012).
The contribution of natural products (NP) to the development of antidiabetic drugs cannot be underestimated (Kazmi et al., 2018). Well-known examples include metformin derived from galegine, the main chemical component of European goat's rue Galega officinalis L., for use in T2DM and dyslipidemias, as well as non-alcoholic fatty liver disease (Hundal and Inzucchi, 2003), and dapagliflozin derived from phlorizin, the main chemical constituent from roots of apple tree Malus pumila Mill., which is the first approved SGLT2 inhibitor for the treatment of T2DM, being an important option in the treatment of diabetes (Dhillon, 2019). Overall, natural products show great potential for the fight against T2DM.
The plants belonging to the genus Hypericum (Hypericaceae) are distributed widely in the temperate and subtropical regions of the northern hemisphere and have been used by folks as a traditional medicine. Polyprenylated acylphloroglucinol derivatives and polyprenylated xanthones with interesting structural characteristics, which catch the attention of chemists, have been isolated from Hypericum genus Xu et al., 2016), and they have various biological activities, such as antidepressant (Verotta, 2003), antimicrobial (Oya et al., 2015), antitumor (Zhang et al., 2014), and hepatoprotective activities (Zhen et al., 2019). Especially studies have shown that ethyl acetate extract of H. perforatum has an antihyperglycemic effect in rats (Arokiyaraj et al., 2011), H. attenuatum regulates dyslipidemia and improves hyperglycemic status (Lv et al., 2019), and H. laricifolium has a good inhibitory effect on α-glucosidase (Quispe et al., 2017). Thus, there is great potential to discover new therapeutics for diabetes from this genus. Hypericum wilsonii N. Robson, which also belongs to the Hypericum genus and has been found to contain 1,2-secohomoadamantane-type polycyclic polyprenylated acylphloroglucinols (PPAPs) (Xie et al., 2020a), is a kind of shrub, growing on hillsides, under forests, or grasslands, at an altitude of 1,000-1750 m. It is mainly distributed in Hubei and Sichuan provinces as well as Chongqing city of China. When we studied the antidiabetic active ingredients of Hypericum wilsonii in vitro, we found that its petroleum ether extract (HW-PE) showed good GLUT4 translocation activity. Bioassay-guided phytochemical investigation on the active HW-PE led to the separation of six new compounds, hyperwilone A (1), hyperwilone B (2), hyperwilone C (3), hyperwilone D (4), wilsonxanthone A (5), and wilsonxanthone B (6), along with 7 known polyprenylated acylphloroglucinol derivatives and a xanthone. Here, we describe the structure elucidation process of the new compounds in detail, the GLUT4 translocation activity in L6 cells of compounds 1-14, and the effects of compound 5 in vitro.

General Experimental Procedures
The 1 H (600 MHz), 13 C (150 MHz), and 2D ( 1 H-1 H COSY, HSQC, HMBC, and ROESY) NMR spectra were recorded on a Bruker Ascend IIITM 600 MHz NMR spectrometer (Bruker Corporation, Fallanden, Switzerland). The HR-ESI-MS data were obtained in the positive ion mode on a UHPLC system and the Q Exactive HF Mass Spectrometer (Thermo Fisher Scientific, United States). The UV spectra were recorded using a UH5300 ultraviolet-visible spectrophotometer (Hitachi, Japan). The IR spectra were measured on a Fourier transform infrared spectrophotometer (Shimadzu, Japan). Fluorescence was measured on a fluorescence microplate reader (Thermo Fisher Scientific, San Jose, CA, United States). Optical rotation was measured using an Autopol IV-T automatic polarimeter (Rudolph Research Analytical, United States). ECD spectra were recorded with a Chirascan Plus circular dichroism spectrometer (Applied Photophysics Ltd., London,

Computational ECD Details
The first step, random searching was used for conformational analyses through the MMFF force field in the Spartan'14. Afterward, the generated conformers were optimized at the B3LYP/6-31G(d) level in Gaussian 09 software with density functional theory (DFT). Time-dependent density functional theory (TD-DFT) method was chosen to calculate the conformers with a Boltzmann population of over 1% at the B3LYP/6-311+G(d, p) level, and SCRF/PCM method was used to evaluate the solvent effects of the MeOH solution. Finally, the Boltzmann-averaged ECD spectra were produced by the SpecDis 1.62 (Bruhn et al., 2013) using a Gaussian band shape with a 0.30 eV exponential half-width. The absolute configurations of 4-6 were resolved by comparing the experiment spectra with the calculated ECD spectra.

NMR Calculations
The conformational analyses process implemented in Spartan'14 was used to search the conformation by using MMFF force field. Gaussian 09 program was used to optimize the geometric structure by DFT at B3LYP/6-31G(d) level, so as to obtain stable conformational isomers. The 13 C NMR chemical shifts were calculated using the PCM solvent continuum model at mPW1PW91/6-311G(d,p) concentration using gaugeindependent atomic orbitals (GIAO) (Grimblat et al., 2015). According to the Boltzmann distribution theory and its relative Gibbs free energy, the average value of the NMR calculated data of the isomers of 1 and 2 is taken. The 13 C NMR chemical shifts of TMS were used as reference by calculating at the same theoretical level. The experimental and computational data of isomers were analyzed by the improved probabilistic DP4+ method (Lodewyk et al., 2012). The higher DP4+ probability score of compounds 1 and 2 indicates that its configuration is correct.

Cell Culture
Rat skeletal muscle L6 cells, obtained from Wuhan Procell Life Science and Technology Co., Ltd, were cultured in complete media containing 1% antibiotics (100 U/mL penicillin and  100 μg/ml streptomycin) (Hyclone, United States), 10% FBS (FBS, Hyclone, United States), and 89% α-MEM (Gibco, United States) at 37°C and in the presence of 5% CO2. The medium was replenished with fresh medium containing 2% FBS when cells was subcultured at the density of 60%, and the medium was replaced every 48 h until the seventh day.

Assay of GLUT4 Translocation
According to the manufacturer's protocol, we used Lipofectamine 2000 to establish an L6 myotube stably overexpressing IRAP-mOrange. IRAP and GLUT4 are two colocalization proteins that exist on GLUT4 storage vesicles (GSV). IRAP can successfully act as a reporter molecule to reflect the transport of GLUT4 protein. IRAP-mOrange-L6 cells were inoculated on sterile coverslips overnight to make the cells completely adherent and then replaced with serum-free α-MEM basic culture media for 2 h to starve cells. Subsequently, we treated the cells with a specific concentration of samples. The images of treated cells were taken by a laser scanning confocal microscope LSM 700 (Carl Zeiss, Jena, Germany) to track the dynamic changes of IRAP-mOrange fluorescence.

Western Blot Analysis
L6 cells were inoculated into a 60-mm dish at a density of 5 × 105 cells and cultured for 1 week. When the cells were coaxed to differentiating into myotubes in α-MEM containing 2% FBS, they were considered suitable for next experiments. Compound C (Calbiochem, San Diego, CA, United States), wortmannin (Selleckchem, Houston, TX, United States), or Go6983 (EMD Millipore, Billerica, MA, United States) was used to pretreat cells for 30 min before treatment with the indicated concentration of compound 5. After 12 h of incubation in a constant temperature incubator, the remaining medium was gently washed off with PBS and the cells were collected in each dish. Then the RIPA protein extraction kit was applied to crack the cells on ice. The parameters of the high-speed centrifuge were set to centrifuge at 15,000 g for 15 min and then the supernatant was collected. The protein concentration of supernatant was quantitated by a bicinchoninic acid (BCA) protein assay kit (Bio-Rad Laboratories, Munich, Germany). After obtaining the concentration and volume of the protein sample, samples were mixed with an appropriate amount of SDS-PAGE protein loading buffer (5×) and denatured in boiling water at 100°C for 10 min. An equivalent amount of samples (30 μg) was loaded on the 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and an electrophoretic separation was performed. Then the protein was electrotransferred to the polyvinylidene difluoride membrane (Pall Corporation, Washington, United States) activated by methanol. The membrane was blocked with 5% skimmed milk for two hours, followed by p-AMPK antibody, AMPK antibody, GLUT4 antibody, and β-actin (Cell Signaling Technology, Danvers, MA, United States) addition and overnight incubation at 4°C. Subsequently, the membranes were incubated with secondary antibodies (Abcam, Cambridge, MA, United States) at appropriate dilution for 2 h. Finally, the protein band attached to a membrane was imaged with an enhanced chemiluminescence (ECL) kit and ChemiDoc XRS (Bio-Rad, California, United States).
Compound 3 was obtained as colorless oil. The molecular formula of hyperwilone C 3) was evidenced to be C 30 1.04-1.73, s). The 13 C NMR data and DEPT (Table 2) indicated that 3 possessed 30 carbons assigned to seven quaternary carbon, four carbonyl, five methine, four methylene, and ten methyl groups. Furthermore, comparing hyperwilone C with wilsonglucinol D by their 1 H, 13 C NMR, and MS data (Xie et al., 2020b) indicated that their spectroscopic data were almost identical, indicating that they were isomers and they possessed the same carbon skeleton. The correlation of H-6β/H-26, H-26/Me-28, and Me-28/H-16 in the ROESY spectrum indicated that H-16 and H-26 were β-orientation, which confirmed that the relative configuration of 3 was the same with wilsonglucinol D. Since CD spectra of 3 showed positive CEs, while wilsonglucinol D showed negative CEs around 330 nm, the absolute configuration of C-1 of 3 was thus determined as S (Xie et al., 2020b) and the absolute configuration of 3 was assigned as (1S,3S,5R,7R,16R,26R).
Thus, the structure of 3 was determined as the enantiomer of wilsonglucinol D and named hyperwilone C.
Compound 6 was obtained as yellow powder. The molecular formula of wilsonxanthone B 6) was evidenced to be C 23 H 22 O 5 by the [M + H] + ion at m/z 379.1539 in HRESIMS (calcd. 379.1545).
Frontiers in Chemistry | www.frontiersin.org September 2021 | Volume 9 | Article 717904 FIGURE 7 | Confocal microscope was used to track IRAP fluorescence changes in L6 cells. L6 cells exposed to the compound 5 for 30 min could significantly induce IRAP fluorescence enhancement.

GLUT4 Translocation Effects of Compounds 1-14
The translocation change of GLUT4 caused by compounds 1-14 could be reflected by the IRAP fluorescence intensity on the L6 cell membrane. After incubating with samples, the IRAP-mOrange fluorescence intensity at the plasma membrane shows varying degrees of change ( Figure 6). Insulin (100 nM) was used as the positive control (PC). Wilsonxanthone A 5) and furohyperforin 8) exerted strong GLUT4 translocation effects, which were enhanced by 1.57 and 1.15 folds, respectively. Hyperwilone A-C (1-3), wilsonxanthone B (6), furoadhyperforin (9), and pseudohenone E (11) show weak to moderate activity with 0.51-to 0.89-fold enhancement, respectively. In Figure 7, compound 5 notably stimulated IRAP fluorescence intensity enhancement after 30 min of exposure to myotubes. This result showed that compound 5 significantly affected GLUT4 translocation in L6 cells. In conclusion, wilsonxanthone A (5), a new xanthone isolated from the aerial parts of Hypericum wilsonii, may possess antidiabetic activity.

Compound 5 Enhanced GLUT4 Translocation and Expression via AMPK Pathway
Previous research has indicated that AMPK, PI3K/Akt, and PKC pathways participated in regulation of GLUT4 transport and expression (Elmendorf, 2002;Thong et al., 2005). Therefore, the inhibitors of corresponding pathways were used to pretreat L6 cells to further explore the mechanism by which compound 5 stimulates GLUT4 expression. Western blotting results show that the effects of GLUT4 increase trigged by compound 5 were totally repressed when compound 5 accompanied by compound C was added ( Figure 8A). In addition, the level of p-AMPK/AMPK and GLUT4 expression was obviously increased following treatment with different concentrations (10, 20, and 30 μg/ml) of compound 5. When the dose of compound 5 was 30 μg/ml, it exerted the strongest stimulatory effect on the translocation of GLUT4 ( Figure 8B). The aforementioned results indicated that compound 5 promotes GLUT4 translocation and expression activation through AMPK pathway in a certain dosedependent manner.

CONCLUSION
In conclusion, fourteen compounds including polyprenylated acylphloroglucinol derivatives and xanthones were isolated from H. wilsonii, in which six compounds were new compounds. Their structures were elucidated on the basis of extensive 1D and 2D NMR spectroscopic data analysis. And the relative configurations and absolute configurations of compounds 1-6 were elucidated based on NMR calculates and comparison of experimental and calculated ECD spectra. Compounds 5 increased GLUT4 translocation and expression via the AMPK pathway. The discovery of new polyprenylated acylphloroglucinol derivatives and xanthones revealed the chemical composition and potentially antidiabetic medicinal value of H. wilsonii.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
JH, TZ, and YM contributed to conception and design of the study. JH and TZ carried out the study and collected important background information. JH, YM, JD, and HC carried out the data acquisition and data analysis. XY, QL, and QW provided assistance for data acquisition, and data analysis. JH and TZ wrote the first draft of the manuscript. XY and HC performed manuscript review. All authors contributed to manuscript revision, read, and approved the submitted version.